A DOUBLE LEUCINE WITHIN THE GLUT4 GLUCOSE-TRANSPORTER COOH-TERMINAL DOMAIN FUNCTIONS AS AN ENDOCYTOSIS SIGNAL

The unique COOH-terminal 30-amino acid region of the adipocyte/skeleta l muscle glucose transporter (GLUT4) appears to be a major structural determinant of this protein's perinuclear localization, from where it is redistributed to the cell surface in response to insulin. To test w hether an underlying mechanism of this domain's function involves gluc ose transporter endocytosis rates, transfected cells were generated ex pressing exofacial hemagglutinin epitope (HA)-tagged erythrocyte/brain glucose transporter (GLUT1) or a chimera containing the COOH-terminal 30 amino acids of GLUT4 substituted onto this GLUT1 construct. Incuba tion of COS-7 or CHO cells expressing the HA-tagged chimera with anti- HA antibody at 37 degrees resulted in an increased rate of antibody in ternalization compared to cells expressing similar levels of HA-tagged GLUT1, which displays a cell surface disposition. Colocalization of t he internalized anti-HA antibody in vesicular structures with internal ized transferrin and with total transporters was established by digita l imaging microscopy, suggesting the total cellular pool of transporte rs are continuously recycling through the coated pit endocytosis pathw ay. Mutation of the unique double leucines 489 and 490 in the rat GLUT 4 COOH-terminal domain to alanines caused the HA-tagged chimera to rev ert to the slow endocytosis rate and steady-state cell surface display characteristic of GLUT1. These results support the hypothesis that th e double leucine motif in the GLUT4 COOH terminus operates as a rapid endocytosis and retention signal in the GLUT4 transporter, causing its localization to intracellular compartments in the absence of insulin.